Several approaches have been used to integrate a DNA of interest into the genome of a plant. In the simplest method, DNA is introduced into a cell and randomly integrates into the genome through illegitimate recombination. One drawback to this method is that positional effects due to random integration make gene expression difficult to analyze.
As an alternative to illegitimate recombination, integration may be targeted to a particular site on the genome through the use of homologous recombination or site-specific recombination. In plants, where homologous recombination technology has not been developed, site-specific recombination is used to integrate a sequence of interest into an integration site that has been previously inserted into the plant host genome. If site-specific integration occurs by a single cross-over event between a chromosome and a circular extrachromosomal replicon, the entire replicon will be inserted into the chromosome. When insertion of the entire replicon is undesirable, a fragment of the replicon comprising the DNA of interest, flanked by target sites for a site-specific recombinase, may be introduced by a double reciprocal cross-over event, into a chromosome having an integration site corresponding to the target sites which flank the DNA of interest. In either case, integration is inefficient because it is reversible, that is, the integrated DNA may be excised by subsequent site-specific recombination between the target sites flanking the integrated DNA.
Several approaches have been taken to avoid excision of an integrated DNA. In one approach, expression of a site-specific recombinase, such as Cre or FLP, is temporally regulated. See O'Gorman et al. (1991) Science 251:1351–1355; Logie and Stewart (1995) Proc. Natl. Acad. Sci. 92:5940–5944; Zhang et al. (1996) Nuc. Acid Res. 24:543–548; Nichols et al. (1997) Mol. Endocinol. 11:950–961; and Feil et al. (1997) Biochem. Biophy. Res. Comm. 237:752–757; the contents of which are incorporated by reference. In these methods, the recombinase is briefly expressed, either transiently or inducibly, in order to allow integration. However, excision of the integrated DNA may occur before active recombinase disappears from the cell. Furthermore, intramolecular excision is kinetically favored over bi-molecular integration. Therefore, integrated DNA is inherently unstable in the presence of recombinase.
A second approach reduces excision of integrated DNA by using pairs of singly mutated target sites on both the chromosome and flanking the DNA of interest. See Albert et al. (1995) Plant J. 7:649–659; Schlake and Bode (1994) Biochemistry 33:12746-12751; O'Gorman et al. (1997) Proc. Natl. Acad. Sci. 94:14602–14607; and Araki et al. (1997) Nuc. Acid Res. 25:868–872; the contents of which are incorporated herein by reference. Recombination between singly mutated target sites results in doubly mutated target sites flanking the DNA inserted into the chromosome. The doubly mutated target sites are not well recognized by the recombinase. Thus, the inserted DNA is excised from the chromosome by a reverse reaction only at low levels. This system, however, has the disadvantage that the singly mutated target sites often do not act as efficient recombination substrates and thus the frequency of integration is reduced. In addition, transformants are unstable because excision may still occur, although at reduced frequency.
Accordingly, it is an object of the invention to provide efficient methods for site-specific integration of DNA into eukaryotic genomes which avoid subsequent excision reactions and other non-productive recombination reactions.